1. Field of the Invention
The present invention generally relates to the field of methods for isolating specific DNA sequences. More particularly, the present invention relates to the field of improved methods of rapid isolation of differentially expressed genes or deleted/inserted sequences in genomic DNA through subtractive hybridization with nucleotide analog-containing subtracter and sequentially enzymatic digestion.
2. Description of the Prior Art
The following references are pertinent to this invention:
1. Bjourson et. al., "Combined Subtraction Hybridization and Polymerase Chain Reaction Amplification Procedure for Isolation of Strain-specific Rhizobium DNA Sequences", Applied and Environmental Microbiology 58: 2296-2301 (1992); PA1 2. Chang et. al., "Cloning and Expression of a Gamma-interferon-inducible Gene in Monocytes: a New Member of a Cytokine Gene Family", International Immunology 1:388-397 (1989); PA1 3. Coochini et. al.,"Identification of Genes Up-regulated in Differentiating Nicotania glauca Pith Tissue, Using an Improved Method for Construction a Subtractive cDNA Library", Nucleic Acids Res. 21: 5742-5747 (1993); PA1 4. Davis et. al., "Expression of a Single Transfected cDNA Converts Fibroblasts to Myoblasts", Cell 51: 987-1000 (1987); PA1 5. Duguid et. al., "Library Subtraction of In Vitro cDNA Libraries to Identify Differentially Expressed Genes in Scapic Infection", Nucleic Acids Res. 18: 2789-2792 (1990); PA1 6. Kunkel et. al., "Specific Cloning of DNA Fragments Absent from the DNA of a Male Patient with an X Chromosome Deletion", Proc. Natl. Acad. Sci. USA 82, 4778-4782 (1985); PA1 7. Lamar et. al., "Y-encoded, Species-specific DNA in Mice: Evidence that the Y Chromosome exists in Two Polymorphic Forms in Inbred Strains," Cell 37:171-177 (1984); PA1 8. Lehninger et. al., "Principles of Biochemistry, 2nd Edition ", Worth Press, pp342-343 (1993); PA1 9. Lisitsyn et. al., "Cloning the Differences Between Two Complex Genomes", Science 259: 946-951 (1993); PA1 10. Littman et. al., "The Isolation and Sequence of the Gene Encoding T8: a Molecule Defining Functional Classes of T Lymphocytes", Cell 40: 237-246 (1985); PA1 11. Maddon et. al., "The Isolation and Nucleotide Sequence of a cDNA Encoding the T Cell Surface Protein T4: a New Member of the Immunoglobulin Gene Family", Cell 42: 93-104 (1985); PA1 12. Nussbaum et. al., "Isolation of Anonymous DNA Sequences from within a Submicroscopic X Chromosomal Deletion in a Patient with Choroideremia, Deafness, and Mental Retardation," Proc. Natl. Acad.Sci. USA 84: 6521-6525 (1987); PA1 13. Sambrook et. al., "Molecular Cloning, 2nd Edition", Cold Spring Harbor Laboratory Press, p10.45 (1989); PA1 14. Straus et. al., "Genomic Subtraction for Cloning DNA Corresponding to Deletion Mutations", Proc. Natl. Acad. Sci. USA 87: 1889-1893 (1990); PA1 15. Wang et. al., "A gene Expression Screen", Proc. Natl. Acad. Sci. USA 88: 11505-11509 (1991); PA1 16. Wicland et. al., "A Method for Difference Cloning; Gene Amplification Following Subtractive Hybridization", Proc. Natl. Acad. Sci. USA 87: 2720-2724 (1990); PA1 17. Ueli et. al., "A Simple and Very Efficient Method for Generating cDNA Libraries", Gene, 25: pp263-269 (1983); and PA1 18. U.S. Pat. No. 5,525,471 issued to Zeng Jin on Jun. 11, 1996 for "Enzymatic Degrading Subtraction Hybridization". PA1 (a) providing a library of nucleotide analog-containing subtracter DNA which is susceptible to the digestion of a nucleotide analog-removing enzyme; PA1 (b) contacting the denatured nucleotide analog-containing subtracter DNA with a library of denatured tester DNA which is not affected by the nucleotide analog-removing enzyme, to form a denatured mixture; PA1 (c) permitting both nucleotide analog-containing subtracter DNA and tester DNA in the denatured mixture under conditions sufficient to form double-stranded hybrid duplexes comprise of homo- and hetero-duplexes; PA1 (d) digesting the nucleotide analog-containing hybrid DNA with the nucleotide analog-removing enzyme to generate abasic-nicks/gaps in the subtracter homoduplex and the subtracter-tester heteroduplex; and PA1 (e) breaking the abasic-nicks/gaps with a single-strand-specific nuclease and thereby provide a library enriched in the library of tester DNA but almost absent in the library of subtracter DNA. PA1 (1) restricting the initial DNA library with a restriction-endonuclease to generate 5'-cohesive termini on both ends; PA1 (2) ligating a specific adaptor to the ends of the restricted DNA where a template is generated for binding with a specific complementary-primer; and PA1 (3) incubating the ligated DNA in denatured form with the specific primer under conditions sufficient to permit the template-dependent extension of the primer to thereby enrich the amount of the initial DNA library and also provide an opportunity for incorporating nucleotide analog into the subtracter in the step (a). PA1 (a') deoxyuridine triphosphate which confers susceptibility to the digestion of uracil-removing enzyme upon incorporation into a DNA molecule; PA1 (b') a specific tester-adaptor/primer which protects both ends of the tester from the digestion of single-strand-specific nuclease, and also confers amplification-capability to the tester DNA; PA1 (c') a specific subtracter-adaptor/primer which is unprotected from the digestion of uracil-removing enzyme, and confers amplification-capability to the uracil-containing subtracter DNA; PA1 (d') a template-dependent dU-incorporation activity; PA1 (e') a uracil-removing enzyme; PA1 (f') a uracil-removing enzyme buffer; PA1 (g') a single-strand-specific nuclease; and PA1 (h') a nuclease buffer.
The ability to compare two different DNA libraries has permitted inquiries into the role of differentially expressed genes or deleted-/inserted-genomic sequences involving the mechanisms of neoplastic transformation, developmental regulation, therapeutic effect, pathological disorder, and cell-physiological phenomena. Understanding the alterations of gene expression and chromosomal rearrangement between normal and disordered cells is especially important for gene therapy, eugenical improvement, pharmaceutical design and etiological investigations.
Several methods have been designed to detect and isolate different DNA sequences which are present in one DNA library but absent in another one. One of the most commonly used methods to accomplish such purpose is subtractive hybridization, involving the elimination of homologous (common) sequences from the mixture of two mutually compared DNA libraries. This kind of selective isolation can be done either between two cDNA libraries (Davis et. al., Cell 51: 987-1000 (1987)), or between two genomic DNA libraries (Lamar et. al., Cell 37: 171-177 (1984)). In brief, this method relies upon the generation of double-stranded DNA libraries from both control cells (subtracter DNA) and cells after treatment, disorder or change (tester DNA). The two DNA libraries are then denatured and hybridized to each other, resulting in subtracter-tester duplex formation if a sequence is common to both DNA populations. By removing the subtracter and common sequence, the remaining DNA is the different sequence which is only present in the tester and also highly related to the treatment, disorder or change of interest.
Subtractive hybridization has been successfully used in the discovery of many functional genes and crucial genomic loci, such as T.sub.4 and T.sub.8 lymphocyte-surface glycoproteins (Maddon et. al., Cell 42: 93-104 (1985); Littman et. al., Cell 40: 237-246 (1985)), gamma-interferon-induced cytokines in monocytes (Chang et. al., International Immunology 1:388-397 (1989)), choroidermia loci (Nussbaum et. al., Proc. Natl. Acad. Sci. USA 84: 6521-6525 (1987)), Duchenne muscular dystrophy-related loci (Kunkel et. al., Proc. Natl. Acad. Sci. USA 82, 4778-4782 (1985)), and human Y-chromosome-specific DNA (Lamar, 1984).
In some cases, the isolated DNA is so abundant in a cell source that it can be detected directly without prior enrichment. In most cases, however, the small amount of desired DNA requires that it be amplified by the polymerase-chain-reaction (PCR), which allows a strengthened observation after multiple cycles of subtractive hybridization (Wang et. al., Proc. Natl. Acad. Sci. USA 88: 11505-11509 (1991); Coochini et. al., Nucleic Acids Res. 21: 5742-5747 (1993)). Additionally, PCR amplification can be used to enrich both subtracter and tester libraries when starting material is limited (Wicland et. al., Proc. Natl. Acad. Sci. USA 87: 2720-2724 (1990)). In short, such amplification is achieved by ligating a specific adaptor to the both ends of an endonuclease-restricted DNA library (amplicon DNA), resulting in the generation of a primer-conjugated region for PCR. However, the PCR amplification may also cause non-specific subtracter contamination when a multiple subtraction/amplification procedure is applied.
Using biotinylation of the subtracter DNA has been widely used to increase subtraction specificity with streptavidin-containing chromatography and to reduce the amount of subtracter needed for hybridization. Straus et. al. (Proc. Natl. Acad. Sci. USA 87: 1889-1893 (1990)) used biotinylated-deletion-mutant genomic DNAs to hybridize with restricted-wild type genomic DNAs, then subtracted the undesired hybrid with avidin-coated beads. The unbound sequences were ligated to specific adaptor and amplified by PCR, resulting in a finding of genomic deletions present in the mutant but absent in the wild type. Meanwhile, Duguid et. al. (Nucleic Acids Res. 18: 2789-2792 (1990)) performed a similar experiment but using a biotinylated double-stranded cDNA library of a normal hamster brain to hybridize with a non-modified cDNA library of a scrapie-infected hamster brain, generating biotinylated complexes which were removed by a biotin-binding avidin resin. The cDNAs remaining in the suspension were amplified and confirmed as scrapie-infected specific gene sequences. Based on experiments like these, it is noteworthy that most previous methods require several cycles of subtractive hybridization because of the incomplete nature of the biotin-avidin affinity interaction. That means: although these methods can successfully reduce the amplification-potential of subtracter DNA, the inevitable use of biotinylation and multiple precipitation/chromatography causes an increase of tedious laboratory-work and a potential loss of desired sequences during repeated subtraction steps.
Bjourson et. al. (Applied and Enviromental Microbiology 58: 2296-2301 (1992)) reported a further improvement in subtractive hybridization methods that employed a biotinylated primer and a uracil-containing deoxynucleotide mixture (e.g. mixture of dATP, dCTP, dGTP and dUTP) to generate uracil-containing DNA (U-DNA) subtracter in PCR. In this case, control and experimental DNA libraries were isolated from different strains of Rhizobium leguminosarum, restricted by an endonuclease, and ligated to different specific adaptors. After that, a special PCR, called uracil-incorporation PCR, was performed to produce the biotinylated subtracter U-DNA which was then hybridized with relatively limited amount of non-modified experimental DNAs, resulting in the formation of biotinylated heterohybrids that contained homologous sequences common to both libraries. Since the biotinylated sequence was removed by streptavidin-phenol-chloroform extraction and the surplus U-DNA was digested with uracil-DNA glycosylase (UDG), the remaining DNAs should be the strain-specific sequences. However, this method still required tedious work in biotinylation and at least two rounds of extraction and chromatography.
Prior art attempts at simplifying subtraction with enzymatic digestion, such as U.S. Pat. No. 5,525,471 to Jin, uses a two-exonuclease degradation precedure. Tester cDNA (from experimental cells) is modified by the incorporation of deoxynucleoside thiotriphosphates which protects the tester from digestion by a first exonuclease. After the tester is hybridized with a non-modified subtracter cDNA (from control cells), the surplus subtracter homohybrid and the entire subtracter-half of the tester-subtracter heterohybrid are digested by the first exonuclease. Before the single-stranded tester half of the heterohybrid can reassociate with each other, a second exonuclease digests all single-stranded tester sequences. This can give a quick, simple way to achieve subtractive hybridization, but it also generates some other problems. First, the property of the desired tester sequence is altered by the modifications which may hinder subsequent analysis. Second, a small amount of reassociation of the single-stranded-tester may occur before the second digestion, resulting in an increase of false-positive results. Third, a long-term, two-exonuclease degradation may damage the small amount of desired sequences, resulting in an increase of false-negative results.
In summary, it is desirable to have a fast, simple, and reliable subtractive hybridization method for distinguishing different sequences from two cDNA or genomic DNA libraries, of which the differences may contribute to developing a therapy for diseases, a diagnosis for inherent problems, or a design for genetic engineering.